Search PubMed⌕ Search

Biomedical subjects

R A Murphy

Publications and source records attributed to R A Murphy.

At least 91 records · Page 5Linked to original sources
PubMed1989

Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle.

Cross-bridge phosphorylation associated with agonist-stimulated contraction of vascular smooth muscle is often transiently elevated. Such observations led to the concept that phosphorylation of the 20-kDa myosin regulatory light chain (Mp) was required for initial activation and cross-bridge cycling but might not be necessary for steady-state maintenance of stress in the latch state. The possibility that stress maintenance is not regulated by phosphorylation has received some experimental support in contractions induced by phorbol esters and the calcium channel activator BAY K 8644 in which significant increases in Mp were not detected. Our aim was to test the hypothesis that phosphorylation is both necessary and sufficient for activation and for maintenance of steady-state stress. Activation of swine carotid media using agents that bypass receptor activation and elevate Ca2+ influx without mobilizing intracellular Ca2+ stores (BAY K 8644 and ionomycin) produced monotonic increases in both stress and Mp. Transient initial peaks in Mp were absent. Steady-state stress induced by both receptor- and nonreceptor-mediated activation was dependent on small increases in Mp. Increases in Mp greater than 0.3 mol Pi/mol myosin light chain had small effects on stress but produced large increases in the maximum rate of cross-bridge cycling at zero load (Vo). The experimentally determined dependence of stress on Mp was quantitatively predicted by our working hypothesis. This model proposes that Ca2+-stimulated cross-bridge phosphorylation is obligatory for cross-bridge attachment. However, dephosphorylation of attached cross bridges to form noncycling "latch bridges" allows stress maintenance with reduced Mp and cycling.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Cross-bridge dephosphorylation and relaxation of vascular smooth muscle.

We tested the hypothesis that relaxation in vascular smooth muscle is the result of inactivation of myosin light chain kinase and cross-bridge dephosphorylation. Fast neurally mediated contractions of swine carotid medial strips were induced by electrical field stimulation. Termination of the stimulus resulted in relaxation with a half time of 2 min. Nifedipine (0.1 microM) increased the relaxation rate without significant effects on the contractile response. Cross-bridge dephosphorylation was much faster than stress decay with basal levels reached within 1 min when 73% of the developed stress remained. The time-course data of dephosphorylation and stress were analyzed with a model that predicted the dependences of stress and isotonic shortening velocity on cross-bridge phosphorylation during contraction. Rate constants resolved from contraction data also fitted the relaxation data when the model's prediction was corrected for estimated errors in the phosphorylation measurements. Because Ca2+-dependent cross-bridge phosphorylation was the only postulated regulatory mechanism in the model, these results are consistent with the hypothesis that cross-bridge dephosphorylation is necessary and sufficient to explain relaxation in the swine carotid media.

Animals↗
PubMed1989

Histamine concentration and Ca2+ mobilization in arterial smooth muscle.

Smooth muscle contraction is dependent on Ca2+ entry from the extracellular space or release from intracellular stores. The sensitivity of these Ca2+ sources to agonist concentration was evaluated by measuring myoplasmic [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in the swine carotid media. High histamine concentrations produced transient elevations in [Ca2+] and phosphorylation with rapid generation of near maximal stress. Lower histamine concentrations produced much smaller [Ca2+] and phosphorylation transients, and stress development was slower. Peak [Ca2+] was proportional to the rate of stress development. Steady-state [Ca2+], phosphorylation, and stress values (which are dependent on extracellular Ca2+) were more sensitive to histamine concentration than was the peak [Ca2+] response both in the presence and absence of extracellular CaCl2 (measures of intracellular Ca2+ release). This result suggests that the mechanism for Ca2+ influx from the extracellular space is more sensitive to histamine than intracellular Ca2+ release. These results are also consistent with the hypothesis that agonist-releasable sarcoplasmic reticular Ca2+ is the major contributor to initial phosphorylation transients that enhance the rate of stress development.

Animals↗
PubMed1989

Expression of nonmuscle myosin heavy and light chains in smooth muscle.

We have compiled evidence that nonmuscle isoforms of both myosin heavy chain (NM MHC) and myosin regulatory light chain (NM LC20) are present in fully differentiated smooth muscles (SM). In swine carotid media sodium dodecyl sulfate-gel electrophoresis separated three MHC bands. The upper two bands were identified by immunoblotting as SM-specific isoforms. The lowest MHC band amounted to 14 +/- 2% of the total MHC and was electrophoretically and antigenically similar to platelet MHC. Two-dimensional gel electrophoresis of swine carotid media extracts resolved multiple LC20 species, including phosphorylated and "satellite" forms. Mass spectrometric analysis of tryptic peptides from blots of these gels demonstrated two LC20 isoforms. The measured peptide masses correspond with two published cDNA sequences proposed to represent SM and NM LC20 isoforms. These sequences readily explain the electrophoretic behavior of the isoforms. The minor isoform's abundance (16 +/- 3%, corresponding to NM MHC), antigenic properties, and pattern of expression in tissue culture all confirm that this is a NM LC20 isoform. The localization and functional significance of NM myosin in smooth muscle is unknown.

Animals↗
PubMed1989

Mechanical properties of carotid arteries from DOCA hypertensive swine.

Carotid arteries from control and deoxycorticosterone acetate (DOCA) hypertensive swine were examined for alterations in structure and in contractile properties. Vessels were excised 7 weeks after subcutaneous implantation of the steroid and subsequent elevation in mean arterial pressure from 102 to 133 mm Hg. The carotid media was 1.8 times thicker in arteries from hypertensive animals than in arteries from control animals. This enlargement was associated with an increase in muscle mass, as the fraction of the media composed of smooth muscle cells remained unchanged. Maximal active stress induced by several agonists normalized for cell cross-sectional area was unaltered. No change was observed in sensitivity or maximal response to norepinephrine, histamine, or KCl depolarization. Isotonic shortening rates were also comparable, as was the time course of shortening velocity to a constant afterload during tonic contractions. It is concluded that an enlargement of the carotid media develops in this model of hypertension. However, this response is not associated with detectable alterations in contractile system function.

Animals↗
PubMed1989

PRO review. Part III: Quality review, intervention, and sanction.

The above material represents a detailed discussion of the GMCF quality review plan, the quality intervention plan, and the sanction plan. It should be clear that every effort is being made by GMCF-PRO to provide fair, realistic, and commonsense quality of care reviews to the physician and hospital community of Georgia. It should also be evident that GMCF interventions will focus on education. Practicing physicians of the appropriate specialty are involved at every level of the review, intervention, and sanction process. Due process is afforded to physicians and hospitals in each plan as described above. The practicing physician community should recognize the obvious need for active participation in the PRO review process to provide proper balance and perspective to quality of care reviews. The GMCF 29-member board (23 physicians) is committed to the concept of maintaining practicing physician involvement and participation in the HCFA-PRO program as mandated by Congressional legislation. It is necessary also for GMCF to continue with close communication to MAG and all specialty societies in order to ensure the recruitment of specialty physician consultants for a quality chart review program, review of screening criteria, and review committee participation.

Georgia↗
PubMed1989

Peer review in the 1990s. A look at the Georgia Medical Care Foundation.

Although it is not possible to completely summarize all of the policies and procedures of a program as complex as the PRO program in one article (or even three), it is useful for establishing the framework of these activities over the next 3 years. Many of us are skeptical of the government's role in the medical review process, and some of us can recall specific instances where problems, e.g., backlogs, have diverted us from our primary mission--to insure quality care. It is imperative, as we begin this new PRO contract, that we all recognize what the PRO program is and what it is not. Specifically, it is not a program of quotas wherein GMCF is required to produce a certain number of denials, quality problems, sanctions, etc. It is a peer review program funded by Medicare but implemented by Georgia physicians with a primary focus on quality review. A second point that needs to be made here concerns the scope of PRO findings relative to the volume of cases reviewed. As with any quality assurance program, it is necessary to look at many cases to determine if there are problems. It is important to note here that GMCF recognizes that most health care delivered in Georgia is appropriate. In fact, Mr. Thomas Morford, National PRO Program Director of the Health Care Financing Administration, recently testified to Congress as follows "clearly, the most important observation thus far is that the PROs have not uncovered any systemic quality problems in the Medicare program.(ABSTRACT TRUNCATED AT 250 WORDS)

Georgia↗
PubMed1988

Two smooth muscle myosin heavy chains differ in their light meromyosin fragment.

Smooth muscle myosin heavy chains [SM1, approximately 205 kilodaltons (kDa), and SM2, approximately 200 kDa] were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Peptide maps of the two heavy chains showed unique patterns. Limited proteolytic cleavage of purified swine stomach myosin was performed by using a variety of proteases to produce the major myosin fragments which were resolved on SDS gels. A single band was obtained for heavy meromyosin in the soluble fraction following chymotrypsin digestion. However, a variable number of bands were observed for light meromyosin fragments in the insoluble fraction after chymotrypsin digestion. Peptide mapping indicated that the two bands observed after short digestion times with chymotrypsin had relative mobility and solubility properties consistent with approximately 100- and 95-kDa light meromyosin (LMM) fragments. These results indicate that the region of difference between SM1 and SM2 lies in the LMM fragment.

Animals↗
PubMed1988

Depression of mechanical function due to active shortening in the chick anterior latissimus dorsi muscle.

1. The depressive effect of shortening on the mechanical properties of the chick anterior latissimus dorsi muscle was assessed by the isovelocity method during tetanic stimulation where the muscle was subjected to a standard conditioning shortening (7% optimum length at approximately 2% maximum shortening velocity, V0) immediately prior to the isovelocity test. Comparison was made to the mechanical properties of non-conditioned (control) contractions. For any given test isovelocity shortening rate, the observed force was always lower if it was preceded by a conditioning shortening. The percentage difference in isovelocity force between conditioned and control contractions was independent of the test shortening velocity. This suggests that shortening lowered the peak isometric force (F0), but did not affect the shape of the force-velocity relation if normalized to a lower F0. 2. Velocity of unloaded shortening, independently measured by the 'slack' method, was unaffected by the conditioning shortening. 3. The magnitude of the force deficit was diminished if the velocity of the conditioning shortening was increased. 4. Recovery of the force deficit was evaluated by allowing a variable period of isometric force redevelopment between the end of the conditioning shortening and the onset of the test isovelocity shortening. The isovelocity force of the conditioned contraction was less than the corresponding control at all times. Complete recovery was not observed with up to 5 s of additional stimulation. However, recovery was observed if the muscle was allowed to relax briefly. 5. Several possible interpretations of our results were considered. Our results are consistent with the hypothesis that the effect of shortening results from non-uniform sarcomere shortening due to a pre-existing heterogeneity of sarcomere strengths. 6. An Appendix describes: (1) how the force-velocity relation would be affected due to the presence of sarcomere strength heterogeneity, and (2) how a model muscle consisting of a heterogeneous population of sarcomeres would be expected to behave following different types of shortenings.

Animals↗
PubMed1988

Cross-bridge phosphorylation and regulation of latch state in smooth muscle.

We have developed a minimum kinetic model for cross-bridge interactions with the thin filament in smooth muscle. The model hypothesizes two types of cross-bridge interactions: 1) cycling phosphorylated cross bridges and 2) noncycling dephosphorylated cross bridges ("latch bridges"). The major assumptions are that 1) Ca2+-dependent myosin phosphorylation is the only postulated regulatory mechanism, 2) each myosin head acts independently, and 3) latch bridges are formed by dephosphorylation of an attached cross bridge. Rate constants were resolved by fitting data on the time courses of myosin phosphorylation and stress development. Comparison of the rate constants indicates that latch-bridge detachment is the rate-limiting step. Model simulations predicted a hyperbolic dependence of steady-state stress on myosin phosphorylation, which corresponded with the experimental observation of high values of stress with low levels of phosphorylation in intact tissues. Model simulations also predicted the experimental observation that an initial phosphorylation transient only accelerates stress development, with no effect on the final steady-state levels of stress. Because the only Ca2+-dependent regulatory mechanism in this model was activation of myosin light chain kinase, these results are consistent with the hypothesis that myosin phosphorylation is both necessary and sufficient for the development of the latch state.

Animals↗
PubMed1988

Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle.

We have proposed a model that incorporates a dephosphorylated "latch bridge" to explain the mechanics and energetics of smooth muscle. Cross-bridge phosphorylation is proposed as a prerequisite for cross-bridge attachment and rapid cycling. Features of the model are 1) myosin light chain kinase and phosphatase can act on both free and attached cross bridges, 2) dephosphorylation of an attached phosphorylated cross bridge produces a noncycling "latch bridge," and 3) latch bridges have a slow detachment rate. This model quantitatively predicts the latch state: stress maintenance with reduced phosphorylation, cross-bridge cycling rates, and ATP consumption. In this study, we adapted A. F. Huxley's formulation of crossbridge cycling (A. F. Huxley, Progr. Biophys. Mol. Biol. 7: 255-318, 1957) to the latch-bridge model to predict the relationship between isotonic shortening velocity and phosphorylation. The model successfully predicted the linear dependence of maximum shortening velocity at zero external load (V0) on phosphorylation, as well as the family of stress-velocity curves determined at different times during a contraction when phosphorylation values varied. The model implies that it is unnecessary to invoke an internal load or multiple regulatory mechanisms to explain regulation of V0 in smooth muscle.

Algorithms↗
PubMed1988

Sr2+ activates cross-bridge phosphorylation and latch state in smooth muscle.

Sr2+ induced myosin phosphorylation and stress development in both skinned and K+-depolarized, Ca2+-depleted, intact swine carotid media. Although higher concentrations of Sr2+ than Ca2+ were required for phosphorylation and stress development, the dependence of stress on phosphorylation was the same in intact tissues. K+ depolarization in the presence of 5 mM Sr2+ produced a transient in phosphorylation (53.2 +/- 5.1% at 1 min, falling to a steady-state value of 21.7 +/- 2.0%) in Ca2+-depleted tissues in which intracellular stores were refilled with Sr2+. Stress developed without a transient (T1/2 = 0.70 min) to a steady state of 89.7 +/- 2.0% of the stress induced by K+ depolarization in the presence of 1.6 mM Ca2+ (K-PSS). Cross-bridge cycling rate as measured by isotonic shortening velocity was proportional to myosin phosphorylation throughout the contraction. When intracellular stores were not refilled with Sr2+, phosphorylation rose to a sustained value of 28.8 +/- 2.7% and stress developed slowly (T1/2 = 2.9 min) to a steady state of 95.9 +/- 1.5% K-PSS-induced stress. Therefore, an initial phosphorylation transient induced by intracellular Sr2+ release only accelerated stress development without significant effects on steady-state stress or phosphorylation (as was true for Ca2+- induced responses). We concluded that Sr2+ substitutes for Ca2+ in phosphorylation and regulation of the latch state in the swine carotid media.

Animals↗
PubMed1988

Dependence of stress on length, Ca2+, and myosin phosphorylation in skinned smooth muscle.

The purpose of this study was to examine the influence of developed stress and tissue length (and presumably filament overlap) on stress hysteresis in skinned vascular smooth muscle. Stress hysteresis is defined as the level of "extra" stress maintained with reduced myosin light chain (MLC) phosphorylation after decreasing the [Ca2+] from a higher to a lower concentration and was postulated to reflect the formation of dephosphorylated, attached, slowly cycling latch bridges in smooth muscle. Detergent skinned tissues of swine carotid media were used for measurement of isometric force and MLC phosphorylation levels. The tissues were either contracted with various [Ca2+] or were first contracted with 7-10 microM Ca2+ and then exposed to a lower [Ca2+]. These two protocols were used at three tissue lengths; 0.7, 1.0, and 1.4 times the optimum length for force development (Lo). The following results were obtained: 1) the order of developed stress was 1.0 greater than 1.4 greater than 0.7 Lo; 2) the Ca2+ sensitivity of developed stress was similar at 0.7 and 1.0 Lo and decreased at 1.4 Lo; 3) the Ca2+ sensitivity and relative magnitude of stress hysteresis were similar at 0.7 and 1.0 Lo with no evidence of stress hysteresis at 1.4 Lo; and 4) the relationship between stress and MLC phosphorylation was affected by tissue length, but the Ca2+ sensitivity of MLC phosphorylation was not. These results suggest that stress maintenance by dephosphorylated crossbridges may be abolished at 1.4 Lo and that the active stress-MLC phosphorylation relationship is altered at long tissue lengths.

Animals↗